Eksperimentalna analiza vezave pomožnih snovi na zdravilno učinkovino : diplomsko delo

Čokolič, Jan

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Title:	Eksperimentalna analiza vezave pomožnih snovi na zdravilno učinkovino : diplomsko delo
Authors:	ID Čokolič, Jan (Author) ID Bren, Urban (Mentor) More about this mentor... ID Zalar, Matja (Comentor)
Files:	UN_Cokolic_Jan_2023.pdf (3,01 MB) MD5: 3D38D5FE688109ABD83A2A6E37979DF9
Language:	Slovenian
Work type:	Bachelor thesis/paper
Typology:	2.11 - Undergraduate Thesis
Organization:	FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:	Število bioloških zdravil na trgu hitro narašča, vendar so zaradi zahtevnih procesov izdelave, le-ta draga, zaradi strukturne zapletenosti in z njo povezanih nestabilnosti pa imajo omejen rok uporabe. Vsako biološko zdravilo tako poleg zdravilne učinkovine, proteina, vsebuje tudi različne pomožne snovi, male organske molekule, ki izboljšujejo njeno stabilnost in podaljšujejo rok uporabe. Pomožne snovi lahko aktivno učinkovino zaščitijo na več načinov, med drugim tudi tako da se nanjo direktno vežejo. Najbolj pogoste skupine pomožnih snovi, ki se uporabljajo v formulaciji biofarmacevtskih proteinov, so površinsko aktivne snovi, sladkorji in aminokisline. V diplomskem delu smo eksperimentalno preučili vezavo najbolj pogostih pomožnih snovi z modelnim biofarmacevtskim proteinom. Uporabili smo modelni protein BSA ter PS20, PS80, ArgHCl in saharozo, ki spadajo med pomožne snovi. Spremljali smo spreminjanje sekundarne strukture s CD spektrometrijo, določevali smo temperaturo razvijanja proteinskih vzorcev z metodo DSC, določevali smo temperaturo agregacije z metodo DLS ter stopnjo oligomerizacije proteinskih vzorcev z metodo SEC-HPLC. Ugotovili smo, da ima dodatek pomožnih snovi viden vpliv na stabilnost proteina v vzorcu. Z dodatkom pomožnih snovi se sekundarna struktura proteina začne spreminjat pri višji temperaturi, prav tako z dodatkom pomožnih snovi narastejo temperature razvijanja. Temperatura agregacije se prav tako zviša z dodatkom pomožnih snovi, delež monomerov v vzorcu pa se z dodatkom pomožnih snovi zmanjša, medtem ko se deleža dimerov in večjih agregatov povečata. Največje spremembe smo zasledili pri vzorcih, ki so poleg proteina BSA vsebovali saharozo, ArgHCl ter PS20 in PS80, katerih koncentracija je bila nad CMC.
Keywords:	protein, pomožne snovi, sekundarna struktura, temperatura razvijanja, temperatura agregacije, oligomerizacija
Place of publishing:	Maribor
Place of performance:	Maribor
Publisher:	[J. Čokolič]
Year of publishing:	2023
Number of pages:	1 spletni vir (1 datoteka PDF (X, 22 f.))
PID:	20.500.12556/DKUM-85258
UDC:	615.015:547.96(043.2)
COBISS.SI-ID:	167105539
Publication date in DKUM:	11.09.2023
Views:	723
Downloads:	124
Metadata:
Categories:	KTFMB - FKKT
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Licences

License:	CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International

Link:	http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:	The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:	23.08.2023

Secondary language

Language:	English
Title:	Experimental analysis of interactions between excipients and active ingredient
Abstract:	The number of biologic drugs on the market is growing rapidly, but they are expensive due to complex manufacturing processes and have a limited shelf life due to their structural complexity and associated instability. Thus, in addition to the active substance, the protein, each formulation also contains various excipients, small organic molecules, that improve the its stability and extend its shelf life. Excipients can protect the active ingredient in several ways, including by binding directly to it. The most common groups of excipients used in the formulation of biopharmaceutical proteins are surfactants, sugars and amino acids. In this thesis, we experimentally investigated the binding of the most common excipients to a model biopharmaceutical protein. We used the model protein BSA and PS20, PS80, ArgHCl and sucrose as excipients. We monitored the change in secondary structure by CD spectrometry, determined the unfolding or melting temperature of protein samples using DSC method, determined the aggregation temperature by DLS method and analyzed the degree of oligomerisation of protein samples using SEC-HPLC. We found that the addition of excipients has a noticeable effect on the stability of the protein in the sample. With the addition of excipients, the secondary structure of the protein starts to disintegrate at a higher temperature, and the melting temperatures also increase with the addition of excipients. Addition of excipients increases the aggregation temperature of BSA and decreases the percentage of monomers in the sample, while the percentages of dimers and larger aggregates increase. The most significant changes were observed in samples containing BSA with addition of sucrose, ArgHCl, PS20 and PS80, which were above the CMC.
Keywords:	protein, excipients, melting temperature, aggregation temperature, oligomerization

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